Osteoarthritis (OA) is a debilitating condition in which articular cartilage is damaged and its mechanical functions (i.e. load support and lubrication), which are dependent on the biphasic behaviors of the tissue, are progressively compromised. Cartilage's fluid phase, its most abundant component, not only acts as a shock absorber and load bearer (due to the viscoelastic coupling between the fluid and solid phases), but also influences cartilage nutrition/metabolism and chondrocyte mechanotransduction. Alterations in this fluid environment have been implicated in OA. While modeling and perfusion studies have provided valuable insight into cartilage's fluid behaviors, there remains an unmet need for real-time in situ measurement of fluid flow in cartilage. In this grant we propose a novel bioimaging approach to measure the cartilage micro-fluidic environment based upon spatiotemporal image correlation spectroscopy (STICS). STICS is a recently developed imaging technique that has been successfully applied to the study of intracellular solute/protein kinetics in vitro. The objective of this study is to develop and standardized STICS imaging for the in situ measurement of both matrix deformation and solute convection in mechanically loaded cartilage. A dual-color confocal STICS approach will be used to quantify the spatial and loading rate dependency of solid matrix and fluid phase behaviors in bovine cartilage plugs subjected to unconfined compression stress-relaxation tests. We will then extend dual-color STICS transport imaging to the quantification of solute convection in human OA cartilage specimens obtained from total knee arthroplasty patients. In which, we hypothesize that load-induced fluid velocity increases with OA is correlated with tissue damage, and indicative of early cartilage degeneration. The proposed in situ quantification of cartilage's fluid environment, given its critical influence on tissue function, represents an innovative tool for studying cartilage's fluid solid interactions. If successful, STICS will be the first bioimaging approach for the direct real-time quantification of cartilage microfluidics and local solid matrix deformations, which are both potent modulators of chondrocyte function. The proposed studies with the novel dual-color STICS approach will yield new quantitative knowledge of the fluid behavior in both normal and diseased cartilage, enabling future investigations of cartilage biomechanics, biology, and the mechanisms of OA initiation and progression.